Application of beta-alanine in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention discloses an application of beta-alanine in preparation of a medicine for treating pulmonary arterial hypertension. In-vivo and in-vitro pharmacological experiments prove that beta-alanine can inhibit lactic acid-mediated histone milk acylation modification and hypoxia-induced phenotypic transformation of PASMCs (Polyaspartic Sulfonate Molecules); the hemodynamics and pulmonary artery vascular remodeling of the PAH mouse are improved. The beta-alanine can inhibit phenotypic transformation of PASMCs and reverse pulmonary artery vascular remodeling which is a key pathological change in the pulmonary hypertension generation process, has huge potential in treatment of pulmonary hypertension, and has good application prospects and high clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of beta-alanine in preparation of a drug for treating pulmonary arterial hypertension. BACKGROUND

[0002] Pulmonary arterial hypertension (PAH) is a progressive disease, mainly manifested as persistent contraction and structural remodeling of pulmonary arteries, and its mechanism involves endothelial dysfunction, abnormal proliferation of smooth muscle cells, inflammatory response and extracellular matrix deposition, eventually leading to increased pulmonary vascular resistance and increased right ventricular load. Epidemiological data shows that pulmonary arterial hypertension affects more than 750 million patients worldwide, of which 80% are in developing countries, and the prevalence rate in people over 65 years old is as high as 10%, much higher than the 1% in the general population. The median survival time of patients with pulmonary arterial hypertension without treatment is only 2.8 years.

[0003] At present, the drug treatment of pulmonary arterial hypertension mainly targets reversible vasoconstriction, and the commonly used drugs are as follows: prostacyclin and its analogues, endothelin receptor antagonists, phosphodiesterase 5 inhibitors and soluble guanylate cyclase stimulators. These drugs can achieve vasodilation through different signal pathways, reduce pulmonary arterial pressure, and improve exercise tolerance and quality of life. However, the above-mentioned drugs have limited direct inhibitory effect on pulmonary vascular remodeling, and the target is relatively single.

[0004] The above-mentioned targeted drugs can reduce pulmonary arterial pressure by dilating blood vessels to improve symptoms and delay disease progression, but long-term treatment has drug resistance, and the inhibitory effect on abnormal proliferation of smooth muscle cells, extracellular matrix deposition and endothelial cell damage and other irreversible structural changes of pulmonary vessels is limited, and it cannot fundamentally inhibit vascular remodeling to treat the disease. After the application of targeted drugs, the 5-year survival rate of patients is only increased to 57%, so there is an urgent need for new drugs and targets for pulmonary arterial hypertension. SUMMARY

[0005] The purpose of the present application is to overcome the defects in the prior art that there is a lack of effective treatment drugs and targets for pulmonary arterial hypertension.

[0006] In order to achieve the above-mentioned purpose, the present application provides application of beta-alanine in preparation of a drug for treating pulmonary arterial hypertension.

[0007] Preferably, the drug concentration of the beta-alanine is 1% to 1.2%.

[0008] Preferably, the beta-alanine can inhibit the proliferation of pulmonary arterial smooth muscle cells induced by hypoxia, and the hypoxia refers to an oxygen concentration lower than 1%.

[0009] Preferably, the β-alanine inhibits the hypoxia-induced contractile conversion of pulmonary artery smooth muscle cells to a synthetic phenotype, wherein hypoxia refers to an oxygen concentration of less than 1%.

[0010] Preferably, the β-alanine inhibits vascular remodeling by competitively suppressing lactate-mediated histone lactylation modification levels.

[0011] Preferably, the dosage form of the drug includes at least one of subcutaneous injection, intravenous injection, tablet, capsule, and suspension.

[0012] Preferably, the pulmonary hypertension includes at least pulmonary hypertension caused by vascular remodeling.

[0013] Preferably, the pulmonary hypertension includes at least one of idiopathic pulmonary hypertension, hereditary pulmonary hypertension, and drug- and toxin-induced pulmonary hypertension.

[0014] The present invention also provides a pharmaceutical composition for preparing a treatment of pulmonary hypertension, the pharmaceutical composition comprising at least β-alanine.

[0015] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention include at least the following: This invention discovers that β-alanine can competitively inhibit lactate-mediated histone lactylation modification, block the pathological phenotypic transformation of pulmonary artery smooth muscle cells, thereby inhibiting vascular wall remodeling and achieving the effect of treating pulmonary hypertension. The β-alanine can be prepared into a drug that can be used to treat diseases such as pulmonary hypertension, hypertension, restenosis after angioplasty, and atherosclerosis caused by vascular remodeling. Attached Figure Description

[0017] Figure 1 The figure shows the results of the inhibitory effect of β-alanine on the proliferation of PASMCs promoted by oxygen concentrations below 1% in vitro.

[0018] Figure 2 The figure shows the results of the inhibitory effect of β-alanine on the migration of PASMCs promoted by oxygen concentrations below 1% in vitro.

[0019] Figure 3 The figure shows the results of qPCR detection of the inhibitory effect of β-alanine on the conversion of PASMCs to the synthetic phenotype induced by oxygen concentrations below 1% in vitro.

[0020] Figure 4Effects of the beta-alanine of the present application on the hemodynamics and cardiac function of mice with pulmonary arterial hypertension induced by SU5416 (20 mg / kg) combined with hypoxia (SuHx): A: representative graph of right ventricular systolic pressure (RVSP), B: statistical graph of right ventricular systolic pressure (RVSP), C: statistical graph of right ventricular hypertrophy index (RV / LV+S).

[0021] Figure 5 Results of HE pathological staining and statistics of lung blood vessel tissue of mice with pulmonary arterial hypertension induced by SU5416 combined with hypoxia (SuHx) for the beta-alanine of the present application.

[0022] Figure 6 Results of the inhibitory effect of histone lactylation modification of lung tissue of mice with pulmonary arterial hypertension induced by SU5416 combined with hypoxia (SuHx) for the beta-alanine of the present application. DETAILED DESCRIPTION

[0023] The technical solutions of the present application are further described below in combination with the drawings and examples. The experimental methods not mentioned otherwise in the present application are all common experimental methods in the art, and the experimental materials not mentioned otherwise in the present application are all conventional experimental materials in the art.

[0024] It should be noted that the beta-alanine of the present application is BETA-alanine, which is purchased from Sangon Biotech, with a CAS number of 107-95-9, and its chemical structural formula is shown as formula I: ; Formula I.

[0025] At present, the targeted drugs for pulmonary arterial hypertension mainly expand blood vessels to improve symptoms and delay disease progression, but still cannot fundamentally inhibit pulmonary vascular remodeling. Existing studies have shown that, in addition to being a metabolic product, lactic acid can also promote the phenotypic transformation of smooth muscle cells by mediating histone lactylation modification, thereby leading to vascular remodeling. The present application proposes to use BETA-alanine to competitively inhibit the level of lactic acid-mediated histone lactylation modification, thereby blocking the pathological phenotypic transformation of smooth muscle cells, so as to achieve the purpose of directly inhibiting vascular remodeling and treating diseases.

[0026] I. Inhibitory effect of beta-alanine on synthetic phenotype transformation of human primary pulmonary arterial smooth muscle cells (hPASMCs) in vitro 1. EdU detection of cell proliferation Experimental method: hPASMCs in logarithmic growth phase were inoculated in 24-well plates, and when the cells were confluent to 60%, the control group was not treated specially, the model group was treated with 1% oxygen concentration hypoxia, and the drug group was treated with 100 mM BETA-alanine under normoxia or hypoxia for 48 h. After 2x EdU solution was added for incubation for 2 h, it was washed, fixed with 4% paraformaldehyde, treated with Triton X-100, and then reacted with EdU Click reaction solution for 30 min. After washing, Hoechst 3342 was used to stain the nucleus for 10 min, and then the cells were washed and photographed. ImageJ was used to count the proportion of EdU to evaluate the cell proliferation.

[0027] Experimental results: As shown in Figure 1 , compared with the model group, BETA-alanine significantly inhibited the proliferation of hPASMCs induced by hypoxia.

[0028] 2. Scratch test to detect cell migration Experimental method: hPASMCs in logarithmic growth phase were inoculated in 6-well plates, and when the cells were confluent to 80%, a 200 μL gun head was used to vertically scratch the bottom of the plate, and then the cells were washed with PBS twice and photographed. Then the medium was replaced with serum-free medium containing or not containing 100 mM BETA-alanine, and the cells were treated under normoxia or hypoxia (1% oxygen concentration) for 12 h, and then photographed again. ImageJ was used to calculate the scratch width to evaluate the cell migration.

[0029] Experimental results: As shown in Figure 2 , compared with the model group, BETA-alanine significantly inhibited the migration of hPASMCs induced by hypoxia.

[0030] 3. qPCR to detect cell proliferation and synthetic gene phenotype Experimental method: hPASMCs in logarithmic growth phase were inoculated in 6-well plates, and when the cells reached 60% confluence, they were treated with 100 mM BETA-alanine under normoxic and hypoxic (1% oxygen concentration) conditions for 48 h, and then the samples were collected. After the culture medium was aspirated, PBS was added and washed twice. Total RNA was extracted from hPASMCs using TRIzol reagent (Accurate Biology), and the extraction process followed the manufacturer's protocol. 1 μg of total RNA was reverse transcribed into cDNA using Evo M-MLV RT Premix (Accurate Biology). Quantitative real-time PCR was performed on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) in a reaction volume of 20 μL containing SYBR Green Master Mix (Applied Biosystems). All reactions were treated with three technical replicates under the following cycle conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene expression levels were normalized to the endogenous reference gene β-actin, and the 2-ΔΔCt method was used for calculation.

[0031] Experimental results: As shown in Figure 3 , compared with the model group, the contraction-related genes MYH11 (contractile protein: encodes smooth muscle myosin heavy chain, involved in cell contraction and tension maintenance), TAGLN (contractile regulatory protein: stabilizes the actin cytoskeleton, promotes smooth muscle cell differentiation and contraction function), ACTA2 (structural framework protein: encodes α-smooth muscle actin, which constitutes the core component of the cell contraction device), and CNN1 (calponin: regulates actin-myosin interaction) in the drug administration group were up-regulated, and the synthesis-related genes CCND1 (cell cycle driver protein: promotes G1 / S phase transition, drives cell proliferation), COL1A1 (matrix structural protein: constitutes type I collagen fibers, provides mechanical support for tissues), TNC (injury response protein: temporarily expressed in tissue remodeling, promotes cell migration and proliferation), and FN1 (adhesion-migration regulatory protein: mediates cell-matrix adhesion, supports cell migration and tissue repair) were down-regulated, indicating that BETA-alanine inhibited the contraction-to-synthesis phenotype transition of hPASMCs induced by hypoxia.

[0032] II. Protective effect of BETA-alanine on SuHx-induced vascular remodeling in mice with PAH 1. Experimental animal grouping, modeling and treatment Experimental method: 30 8-week-old male C57BL / 6 mice were randomly divided into 3 groups: normoxia group, hypoxia model group, and hypoxia administration group. The mice were placed in a hypoxic chamber (10% oxygen concentration) for four weeks, and subcutaneous injection of SU5416 (20 mg / kg) was performed once a week to induce a mouse pulmonary hypertension model. The administration group replaced the normal drinking water with 200 mL of 1.2% BETA-alanine drinking water two weeks after hypoxia modeling, and replaced it once a week, and gavaged 200 μL of 1.2% BETA-alanine aqueous solution 3 times a week, until the end of the experiment.

[0033] The hypoxia model group of the animal experiment was developed into pulmonary hypertension by exposing the mice to a hypoxic (10% oxygen concentration) environment for 3-4 weeks and injecting a VEGF receptor inhibitor SU5416 once at the beginning of the experiment. Therefore, hypoxia combined with SU5416 is a necessary condition for this pulmonary hypertension modeling method 2. Hemodynamic detection Right ventricular pressure measurement: After the mice were anesthetized with tribromoethanol, the chest wall was carefully cut to expose the heart, the tip of a 1.2F micro pressure sensor catheter was inserted into the right ventricle, and the PowerLab data acquisition system was used to stably and continuously detect the right ventricular pressure value of the mouse.

[0034] Cardiac function measurement: PBS was used to wash the lung tissue from the right ventricle until it turned white, the mouse ventricle was carefully cut off, the mouse right ventricle, left ventricle and interventricular septum were separated and weighed, the RV / LV+S value was calculated and counted.

[0035] 3. HE pathological staining The mouse lung tissue was fixed with 4% paraformaldehyde for 24 hours, then paraffin-embedded, sectioned and H&E stained.

[0036] Experimental results: As shown in Figure 4 , the right ventricular systolic pressure of the hypoxia administration group of mice was reduced, indicating that the pulmonary arterial pressure was reduced compared with the model group; the right ventricular hypertrophy index (RV / LV+S) was reduced, indicating that the right ventricular hypertrophy caused by increased pulmonary arterial pressure was also relieved; as shown in Figure 5 , HE staining showed that the pulmonary arterial blood vessels were thin, indicating that BETA-alanine reduced the thickening of the pulmonary arterial wall.

[0037] 4. Western Blot detection of the inhibitory effect of BETA-alanine on SU5416+hypoxia (SuHx) induced pulmonary hypertension in mouse lung tissue histone acylation modification Experimental method: 20 mg of mouse lung tissue and two grinding beads were put into each grinding tube, 100 mg / 1 mL of pre-cooled RIPA strong lysis solution and 100x protease inhibitor were added, 50hz grinding for 3 min, lysis for 10 min on ice, centrifugation at 12000 rpm, and the supernatant was collected. After BCA quantification, loading buffer was added, boiled at 100℃ for 10 min, and stored at-20℃. The proteins were separated using a 15% SDS-PAGE gel, transferred to a 0.2 μm pore size PVDF membrane, blocked with 5% skim milk at room temperature for 1.5 h, and then incubated with specific primary antibodies overnight. The next day, the HRP-labeled secondary antibody was incubated at room temperature for 1.5 h, and then developed using an AI600 color development system.

[0038] Experimental results: As shown in Figure 6 , the level of histone lactylation modification caused by the hypoxia model in the lung tissue of the hypoxia administration group of mice was significantly reduced compared with the hypoxia model group. Histone lactylation modification, as a new epigenetic modification, is reported to widely regulate the expression of proliferation-related genes in PASMCs in a hypoxic pulmonary hypertension model.

[0039] In vitro studies show that BETA-alanine significantly inhibits the phenotypic transformation and proliferation of pulmonary artery smooth muscle cells induced by hypoxia. Animal experiments show that BETA-alanine administration significantly improves the thickening of the pulmonary artery wall, a key pathological process in the development of pulmonary hypertension, by inhibiting the level of lactate-mediated histone lactylation modification in the hypoxia model, reduces the right ventricular systolic pressure, i.e. pulmonary artery pressure, and reduces the right ventricular hypertrophy caused by pulmonary hypertension. In summary, BETA-alanine can treat pulmonary hypertension by directly inhibiting the important pathological process of vascular remodeling of smooth muscle cells.

[0040] BETA-alanine is a non-essential amino acid that can increase the content of carnosine in skeletal muscle as a sports nutrition, improve sports performance, has high safety, and the raw material is easy to obtain. In vivo and in vitro efficacy tests have verified that BETA-alanine inhibits lactate-mediated histone lactylation modification and hypoxia-induced PASMCs phenotypic transformation, and improves the hemodynamics and pulmonary artery remodeling of pulmonary hypertension mice. In summary, BETA-alanine can inhibit the phenotypic transformation of PASMCs and reverse the pulmonary artery remodeling, a key pathological change in the development of pulmonary hypertension. It has great potential in the treatment of pulmonary hypertension and good application prospects and high clinical application value.

[0041] Similarly, the present application also provides a pharmaceutical composition for treating pulmonary hypertension, which comprises BETA-alanine.

[0042] In summary, the application provides application of beta-alanine in preparation of a drug for treating pulmonary arterial hypertension, and the application verifies that beta-alanine can inhibit lactic acid-mediated histone lactylation modification and hypoxia-induced phenotype conversion of PASMCs through in-vivo and in-vitro pharmacodynamic experiments, and improves hemodynamics and pulmonary arterial vascular remodeling of PAH mice. The beta-alanine can inhibit phenotype conversion of PASMCs and reverse pulmonary arterial vascular remodeling, which is a key pathological change in the occurrence process of pulmonary arterial hypertension, and has great potential in treating pulmonary arterial hypertension, and has good application prospect and high clinical application value.

[0043] Although the present application has been described in detail by the preferred embodiments above, it should be appreciated that the above description should not be considered as limiting the present application. Various modifications and substitutions to the present application will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present application should be defined by the appended claims.

Claims

1. Application of β-alanine in the preparation of drugs for treating pulmonary hypertension.

2. The application as described in claim 1, characterized in that, The drug concentration of β-alanine is 1% to 1.2%.

3. The application as described in claim 1, characterized in that, The β-alanine can inhibit the proliferation of pulmonary artery smooth muscle cells induced by hypoxia, where hypoxia refers to an oxygen concentration of less than 1%.

4. The application as described in claim 1, characterized in that, The β-alanine inhibits the hypoxia-induced contractile conversion of pulmonary artery smooth muscle cells to a synthetic phenotype, where hypoxia refers to an oxygen concentration below 1%.

5. The application as described in claim 1, characterized in that, The β-alanine inhibits vascular remodeling by competitively suppressing lactate-mediated histone lactylation modification levels.

6. The application as described in claim 1, characterized in that, The dosage form of the drug includes at least one of the following: subcutaneous injection, intravenous injection, tablets, capsules, and suspension.

7. The application as described in claim 1, characterized in that, The pulmonary hypertension includes at least pulmonary hypertension caused by vascular remodeling.

8. The application as described in claim 1, characterized in that, The pulmonary hypertension includes at least one of idiopathic pulmonary hypertension, hereditary pulmonary hypertension, and drug- and toxin-induced pulmonary hypertension.

9. A pharmaceutical composition for preparing a treatment of pulmonary hypertension, characterized in that, The pharmaceutical composition contains at least β-alanine.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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